Cotton event PV-GHGT07(1445) and compositions and methods for detection thereof

ABSTRACT

The present invention provides DNA compositions and assays for detecting the presence of the DNA compositions in PV-GHGT07(1445) cotton event based on the DNA sequence of the recombinant construct inserted into the cotton genome and of the genomic sequences flanking the insertion site. Kits and conditions useful in conducting the assays are provided.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/243,190, filed Oct. 25, 2000, herein incorporated byreference in its entirety.

BACKGROUND OF INVENTION

[0002] The present invention relates to the field of plant molecularbiology, more specifically the invention relates to transgenicglyphosate tolerance in a plant. The invention more specifically relatesto a glyphosate tolerant cotton plant PV-GHGT07(1445) and to assays fordetecting the presence of cotton plant PV-GHGT07(1445) DNA in a sampleand compositions thereof.

[0003] Cotton is an important fiber crop in many areas of the world. Themethods of biotechnology have been applied to cotton for improvement ofthe agronomic traits and the quality of the product. The method ofintroducing transgenes into cotton plants is demonstrated in U.S. Pat.No. 5,004,863. One such agronomic trait important in cotton productionis herbicide tolerance, in particular, tolerance to glyphosateherbicide. This trait has been introduced into cotton plants and is asuccessful product now used in cotton production. The expression offoreign genes in plants is known to be influenced by their chromosomalposition, perhaps due to chromatin structure (e.g., heterochromatin) orthe proximity of transcriptional regulation elements (e.g., enhancers)close to the integration site (Weising et al., Ann. Rev. Genet22:421-477, 1988). For this reason, it is often necessary to screen alarge number of events in order to identify an event characterized byoptimal expression of a introduced gene of interest. For example, it hasbeen observed in plants and in other organisms that there may be a widevariation in levels of expression of introduced genes among events.There may also be differences in spatial or temporal patterns ofexpression, for example, differences in the relative expression of atransgene in various plant tissues, that may not correspond to thepatterns expected from transcriptional regulatory elements present inthe introduced gene construct. For this reason, it is common to producehundreds to thousands of different events and screen those events for asingle event that has desired transgene expression levels and patternsfor commercial purposes. An event that has desired levels or patterns oftransgene expression is useful for introgressing the transgene intoother genetic backgrounds by sexual outcrossing using conventionalbreeding methods. Progeny of such crosses maintain the transgeneexpression characteristics of the original transformant. This strategyis used to ensure reliable gene expression in a number of varieties thatare well adapted to local growing conditions.

[0004] It would be advantageous to be able to detect the presence of aparticular event in order to determine whether progeny of a sexual crosscontain a transgene of interest. In addition, a method for detecting aparticular event would be helpful for complying with regulationsrequiring the premarket approval and labeling of foods derived fromrecombinant crop plants, for example. It is possible to detect thepresence of a transgene by any well known nucleic acid detection methodsuch as the polymerase chain reaction (PCR) or DNA hybridization usingnucleic acid probes. These detection methods generally focus onfrequently used genetic elements, such as promoters, terminators, markergenes, etc. As a result, such methods may not be useful fordiscriminating between different events, particularly those producedusing the same DNA construct unless the sequence of chromosomal DNAadjacent to the inserted DNA (“flanking DNA”) is known. Anevent-specific PCR assay is discussed, for example, by Windels et al.(Med. Fac. Landbouww, Univ. Gent 64/5b:459-462, 1999), who identifiedglyphosate tolerant soybean event 40-3-2 by PCR using a primer setspanning the junction between the insert and flanking DNA, specificallyone primer that included sequence from the insert and a second primerthat included sequence from flanking DNA.

[0005] This invention relates to the glyphosate herbicide tolerantcotton (Gossypium hirsutum) plant PV-GHGT07(1445) sold in the U.S.A. andother countries under the name of Roundup Ready® cotton and to the DNAmolecules contained in these cotton plants that are useful in detectionmethods of Roundup Ready® cotton and progeny thereof.

SUMMARY OF INVENTION

[0006] According to an aspect of the invention, compositions and methodsare provided for detecting the presence of the transgene/genomicinsertion region from a cotton plant designated PV-GHGT07(1445) plantsand seeds. DNA sequences are provided that comprise at least onetransgene/genomic insertion region junction sequence of PV-GHGT07(1445)identified as SEQ ID NO:5 and SEQ ID NO:6, and complements thereof;wherein an insertion region junction sequence spans the junction betweenheterologous DNA inserted into the genome and the DNA from the cottoncell flanking the insertion site and is diagnostic for the event.

[0007] According to another aspect of the invention, DNA sequences thatcomprise the novel transgene/genomic insertion region, SEQ ID NO:7 arean aspect of this invention. Included are DNA sequences that comprise asufficient length of polynucleotides of transgene insert sequence and asufficient length of polynucleotides of cotton genomic sequence fromcotton plant PV-GHGT07(1445) of SEQ ID NO:7 that are useful as primersequences for the production of an amplicon product diagnostic forcotton plant PV-GHGT07(1445).

[0008] According to another aspect of the invention, DNA sequences thatcomprise the novel transgene/genomic insertion region, SEQ ID NO:8 arean aspect of this invention. Included are DNA sequences that comprise asufficient length of polynucleotides of transgene insert sequence and asufficient length of polynucleotides of cotton genomic sequence fromcotton plant PV-GHGT07(1445) of SEQ ID NO:8 that are useful as primersequences for the production of an amplicon product diagnostic forcotton plant PV-GHGT07(1445).

[0009] According to another aspect of the invention, the DNA sequencesthat comprise at least 11 or more nucleotides of the transgene portionof the DNA sequence of SEQ ID NO:7 or complements thereof, and a similarlength of 5′ flanking cotton DNA sequence of SEQ ID NO:7 or complementsthereof are useful as DNA primers in DNA amplification methods. Theamplicons produced using these primers are diagnostic for cotton eventPV-GHGT07(1445). Therefore the invention also includes the ampliconsproduced by DNA primers homologous or complementary to SEQ ID NO:7.

[0010] According to another aspect of the invention, the DNA sequencesthat comprise a sufficient length of polynucleotides of the transgeneportion of the DNA sequence of SEQ ID NO:8 or complements thereof, and asimilar length of 5′ flanking cotton DNA sequence of SEQ ID NO:8 orcomplements thereof are useful as DNA primers in DNA amplificationmethods. The amplicons produced using these primers are diagnostic forcotton event PV-GHGT07(1445). Therefore the invention also includes theamplicons produced by DNA primers homologous or complementary to SEQ IDNO:7.

[0011] According to another aspect of the invention, methods ofdetecting the presence of DNA corresponding to the cotton eventPV-GHGT07(1445) event in a sample are provided. Such methods comprise:(a) contacting the sample comprising DNA with a primer set that, whenused in a nucleic acid amplification reaction with DNA from cotton eventPV-GHGT07(1445), produces an amplicon that is diagnostic for cottonevent PV-GHGT07(1445); (b) performing a nucleic acid amplificationreaction, thereby producing the amplicon; and (c) detecting theamplicon.

[0012] According to another aspect of the invention, methods ofdetecting the presence of a DNA corresponding to the PV-GHGT07(1445)event in a sample are provided, such methods comprising: (a) contactingthe sample comprising DNA with a probe that hybridizes under stringenthybridization conditions with DNA from cotton event PV-GHGT07(1445) anddoes not hybridize under the stringent hybridization conditions with acontrol cotton plant (non-PV-GHGT07(1445) DNA); (b) subjecting thesample and probe to stringent hybridization conditions; and (c)detecting hybridization of the probe to the DNA.

[0013] According to another aspect of the invention, methods ofproducing a cotton plant that tolerates application of glyphosate areprovided that comprise the steps of: (a) sexually crossing a firstparental cotton line comprising the expression cassettes of the presentinvention, which confers tolerance to application of glyphosate, and asecond parental cotton line that lacks the glyphosate tolerance, therebyproducing a plurality of progeny plants; and (b) selecting a progenyplant by the use of molecular markers SEQ ID NO:5 and SEQ ID NO:6. Suchmethods may optionally comprise the further step of back-crossing theprogeny plant to the second parental cotton line to producing atrue-breeding cotton plant that tolerates application of glyphosate.

[0014] According to another aspect of the invention, methods ofdetermining the zygosity of progeny of a cross with PV-GHGT07(1445) areprovided. A method that comprises contacting a sample consisting ofcotton DNA with a primer set comprising SEQ ID NO:9, SEQ ID NO:11 andSEQ ID NO:12, that when used in a nucleic-acid amplification reactionwith genomic DNA from cotton event PV-GHGT07(1445), produces a firstamplicon that is diagnostic for cotton event PV-GHGT07(1445); andperforming a nucleic acid amplification reaction, thereby producing thefirst amplicon; and detecting the first amplicon; and contacting thesample comprising cotton DNA with said primer set, that when used in anucleic-acid amplification reaction with genomic DNA from cotton plantsproduces a second amplicon comprising the native cotton genomic DNAhomologous to the cotton genomic region of a transgene insertionidentified as cotton event PV-GHGT07(1445); and performing a nucleicacid amplification reaction, thereby producing the second amplicon; anddetecting the second amplicon; and comparing the first and secondamplicons in a sample, wherein the presence of both amplicons indicatesthe sample is heterozygous for the transgene insertion. The foregoingand other aspects of the invention will become more apparent from thefollowing detailed description.

DETAILED DESCRIPTION

[0015] The following definitions and methods are provided to betterdefine the present invention and to guide those of ordinary skill in theart in the practice of the present invention. Unless otherwise noted,terms are to be understood according to conventional usage by those ofordinary skill in the relevant art. Definitions of common terms inmolecular biology may also be found in Rieger et al., Glossary ofGenetics: Classical and Molecular, 5th edition, Springer-Verlag: NewYork, 1991; and Lewin,—Genes V, Oxford University Press: New York, 1994.The nomenclature for DNA bases as set forth at 37 CFR § 1.822 is used.

[0016] As used herein, the term “cotton” means Gossypium hirsutum andincludes all plant varieties that can be bred with cotton, includingwild cotton species.

[0017] As used herein, the term “comprising” means “including but notlimited to”.

[0018] “Glyphosate” refers to N-phosphonomethylglycine and its salts.Glyphosate is the active ingredient of Roundup® herbicide (MonsantoCo.). Treatments with “glyphosate herbicide” refer to treatments withthe Roundups, Roundup Ultra® herbicide or any other herbicideformulation containing glyphosate. For the purposes of the presentinvention, the term “glyphosate”includes any herbicidally active form ofN-phosphonomethylglycine (including any salt thereof) and other formsthat result in the production of the glyphosate anion in plants.Treatments with “glyphosate” refer to treatments with the Roundups orRoundup Ultra® herbicide formulation, unless otherwise stated. Planttransformation and regeneration in tissue culture use glyphosate orsalts of glyphosate. Whole plant assays use formulated Roundups orRoundup Ultra®. Additional formulations with herbicide activity thatcontain N-phosphonomethylglycine or any of its salts are herein includedas a glyphosate herbicide.

[0019] A transgenic “event” is produced by transformation of plant cellswith heterologous DNA, i.e., a nucleic acid construct that includes atransgene of interest, regeneration of a population of plants resultingfrom the insertion of the transgene into the genome of the plant, andselection of a particular plant characterized by insertion into aparticular genome location. The term “event” refers to the originaltransformant and progeny of the transformant that include theheterologous DNA. The term “event” also refers to progeny produced by asexual outcross between the transformant and another variety thatincludes the genomic/transgene DNA. Even after repeated back-crossing toa recurrent parent, the inserted transgene DNA and flanking genomic DNA(genomic/transgene DNA) from the transformed parent is present in theprogeny of the cross at the same chromosomal location. The term “event”also refers to DNA from the original transformant and progeny thereofcomprising the inserted DNA and flanking genomic sequence immediatelyadjacent to the inserted DNA that would be expected to be transferred toa progeny that receives inserted DNA including the transgene of interestas the result of a sexual cross of one parental line that includes theinserted DNA (e.g., the original transformant and progeny resulting fromselfing) and a parental line that does not contain the inserted DNA.

[0020] A glyphosate tolerant cotton plant can be bred by first sexuallycrossing a first parental cotton plant consisting of a cotton plantgrown from PV-GHGT07(1445) seed (also referred to as event 1445) thattolerates application of glyphosate herbicide, and a second parentalcotton plant that lacks the tolerance to glyphosate herbicide, therebyproducing a plurality of first progeny plants; and then selecting afirst progeny plant that is tolerant to application of glyphosateherbicide; and selfing the first progeny plant, thereby producing aplurality of second progeny plants; and then selecting from the secondprogeny plants a glyphosate herbicide tolerant plant. These steps canfurther include the back-crossing of the first glyphosate tolerantprogeny plant or the second glyphosate tolerant progeny plant to thesecond parental cotton plant or a third parental cotton plant, therebyproducing a cotton plant that tolerates the application of glyphosateherbicide. A cotton crop comprising cotton seeds PV-GHGT07(1445) orprogeny thereof can be planted in a field and treated with a sufficientamount of glyphosate herbicide to control the weeds withoutsignificantly affecting the cotton crop. A sufficient amount ofglyphosate herbicide is about 8 ounces/acre or more, 16 ounces/acre ormore, 32 ounces/acre or more, or 64 ounces/acre or more. Any glyphosatecontaining herbicide formulation can be used to control weeds in acotton crop comprising PV-GHGT07(1445) plants or progeny thereof.

[0021] It is also to be understood that two different transgenic plantscan also be mated to produce offspring that contain two independentlysegregating added, exogenous genes. Selfing of appropriate progeny canproduce plants that are homozygous for both added, exogenous genes.Back-crossing to a parental plant and out-crossing with a non-transgenicplant are also contemplated, as is vegetative propagation. Descriptionsof other breeding methods that are commonly used for different traitsand crops can be found in one of several references, e.g., Fehr,in—Breeding Methods for Cultivar Development, Wilcox J. ed., AmericanSociety of Agronomy, Madison Wis. (1987) herein incorporated byreference in its entirety; Poehlman, J. M. (1987); Breeding Field Crops,3rd ed. Van Nostrand Reinhold, NY, Knott, D. R. (1987); hereinincorporated by reference in its entirety. Backcross breeding has beenused to transfer genes for a simply inherited, highly heritable traitinto a desirable homozygous cultivar or inbred line, which is therecurrent parent. The source of the trait to be transferred is calledthe donor parent. The resulting plant is expected to have the attributesof the recurrent parent (e.g., cultivar) and the desirable traittransferred from the donor parent. After the initial cross, individualspossessing the phenotype of the donor parent are selected and repeatedlycrossed (backcrossed) to the recurrent parent. The resulting parent isexpected to have the attributes of the recurrent parent (e.g., cultivar)and the desirable trait transferred from the donor parent.

[0022] The DNA molecules of the present invention can by used asmolecular markers in a marker assisted breeding (MAB) method. DNAmolecules of the present invention can be used in methods, such as, AFLPmarkers, RFLP markers, RAPD markers, SNPs, and SSRs that identifygenetically linked agronomically useful traits as described by Walton,Seed World 22-29 (July, 1993), the entirety of which is hereinincorporated by reference; Burow and Blake, Molecular Dissection ofComplex Traits, 13-29, Eds. Paterson, CRC Press, New York (1988), theentirety of which is herein incorporated by reference). The glyphosatetolerance trait can be tracked in the progeny of a cross with cottonplant PV-GHGT07(1445) plants or progeny thereof and any other cottoncultivar or variety using the MAB methods. The DNA molecules are markersfor this trait and in MAB methods that are well known in the art can beused to track glyphosate tolerance in cotton where PV-GHGT07(1445)plants or progeny thereof was a parent or ancestor.

[0023] Commercial glyphosate cotton varieties containinggenomic/transgene DNA from cotton event PV-GHGT07(1445) are known asRoundup Ready® cotton and have been introduced and are available in atleast the following varieties: Acala Riata RR, DP 409 B/RR, DP 420 RR,DP 422 B/RR, DP 425 RR, DP 429 RR, DP 436 RR, DP 450 B/RR, DP 451 B/RR,DP 458 B/RR, DP5415 RR, DP 5690 RR DP 6100 RR Acala, DP 655 B/RR, DP 90RR, DP 9834 B.RR, PM 1215 BG/RR, PM 1218 BG/RR, PV 1220 BG/RR, PM 1244BG/RR, PM 1560 BG/RR, PM 2145 RR, PM 2156 RR, PM 2200 RR, PM 2280 BG/RR,PM 2326 RR, PM 2326 BG/RR, PM 2320 RR, PM 2379 RR, ST 4892 BR, SG 125BR, SG 125 R, SG 150 BR, SG 150 R, SG 501 BR, SG 521 BR, and SG 521 R.These glyphosate tolerant cotton varieties and any glyphosate tolerantcotton variety derived from these varieties represent the progeny of thecotton event PV-GHGT07(1445). The methods of the (present invention canbe used to identify any glyphosate tolerant cotton variety that is aprogeny of cotton event PV-GHGT07(1445).

[0024] A “probe” is an isolated nucleic acid to which is attached aconventional detectable label or reporter molecule, e.g., a radioactiveisotope, ligand, chemiluminescent agent, or enzyme. Such a probe iscomplementary to a strand of a target nucleic acid, in the case of thepresent invention, to a strand of genomic DNA from cotton eventPV-GHGT07(1445) whether from a cotton plant or from a sample thatincludes DNA from the event. Probes according to the present inventioninclude not only deoxyribonucleic or ribonucleic acids but alsopolyamides and other probe materials that bind specifically to a targetDNA sequence and can be used to detect the presence of that target DNAsequence.

[0025] “Primers” are isolated nucleic acids that are annealed to acomplementary target DNA strand by nucleic acid hybridization to form ahybrid between the primer and the target DNA strand, then extended alongthe target DNA strand by a polymerase, —e.g., a DNA polymerase. Primerpairs of the present invention refer to their use for amplification of atarget nucleic acid sequence, e.g., by the polymerase chain reaction(PCR) or other conventional nucleic-acid amplification methods.

[0026] Probes and primers are generally 8 polynucleotides or more inlength, 18 polynucleotides or more, 24 polynucleotides or more, or 30polynucleotides or more. Such probes and primers hybridize specificallyto a target sequence under high stringency hybridization conditions.Preferably, probes and primers according to the present invention havecomplete sequence similarity with the target sequence, although probesdiffering from the target sequence and that retain the ability tohybridize to target sequences may be designed by conventional methods.

[0027] Methods for preparing and using probes and primers are described,for example, in Molecular Cloning: A Laboratory Manual, 2nd ed., vol.1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989 (hereinafter, “Sambrook et al., 1989”), hereinincorporated by reference in its entirety; Current Protocols inMolecular Biology, ed. Ausubel et al., Greene Publishing andWiley-Interscience, New York, 1992 (with periodic updates) (hereinafter,“Ausubel et al., 1992”), herein incorporated by reference in itsentirety; and Innis et al., PCR Protocols: A Gulde to Methods andApplications, Academic Press: San Diego, 1990, herein incorporated byreference in its entirety. PCR-primer pairs can be derived from a knownsequence, for example, by using computer programs intended for thatpurpose such as Primer (Version 0.5, ©1 991, Whitehead Institute forBiomedical Research, Cambridge, Mass.), herein incorporated by referencein its entirety.

[0028] Primers and probes based on the flanking DNA and insert sequencesdisclosed herein can be used to confirm (and, if necessary, to correct)the disclosed sequences by conventional methods, e.g., by re-cloning andsequencing such sequences.

[0029] The nucleic acid probes and primers of the present inventionhybridize under stringent conditions to a target DNA sequence. Anyconventional nucleic acid hybridization or amplification method can beused to identify the presence of DNA from a transgenic event in asample. Nucleic acid molecules or fragments thereof are capable ofspecifically hybridizing to other nucleic acid molecules under certaincircumstances. As used herein, two nucleic acid molecules are said to becapable of specifically hybridizing to one another if the two moleculesare capable of forming an anti-parallel, double-stranded nucleic acidstructure. A nucleic acid molecule is said to be the “complement” ofanother nucleic acid molecule if they exhibit complete complementarity.As used herein, molecules are said to exhibit “complete complementarity”when every nucleotide of one of the molecules is complementary to anucleotide of the other. Two molecules are said to be “minimallycomplementary” if they can hybridize to one another with sufficientstability to permit them to remain annealed to one another under atleast conventional “low-stringency” conditions. Similarly, the moleculesare said to be “complementary” if they can hybridize to one another withsufficient stability to permit them to remain annealed to one anotherunder conventional “high-stringency” conditions. Conventional stringencyconditions are described by Sambrook et al., 1989, and by Haymes et al.,In: Nucleic Acid Hybridization, A Practical Approach, IRL Press,Washington, D.C. (1985), herein incorporated by reference in itsentirety. Departures from complete complementarity are thereforepermissible, as long as such departures do not completely preclude thecapacity of the molecules to form a double-stranded structure. In orderfor a nucleic acid molecule to serve as a primer or probe it need onlybe sufficiently complementary in sequence to be able to form a stabledouble-stranded structure under the particular solvent and saltconcentrations employed.

[0030] As used herein, a substantially homologous sequence is a nucleicacid sequence that will specifically hybridize to the complement of thenucleic acid sequence to which it is being compared under highstringency conditions. The term “stringent conditions” is functionallydefined with regard to the hybridization of a nucleic-acid probe to atarget nucleic acid (i.e., to a particular nucleic-acid sequence ofinterest) by the specific hybridization procedure discussed in Sambrooket al., 1989, at 9.52-9.55.—See also, Sambrook et al., 1989 at9.47-9.52, 9.56-9.58 herein incorporated by reference in its entirety;Kanehisa, (Nucl. Acids Res. 12:203-213, 1984, herein incorporated byreference in its entirety); and Wetmur and Davidson, (1. Mol. Biol.31:349-370, 1988, herein incorporated by reference in its entirety).Accordingly, the nucleotide sequences of the invention may be used fortheir ability to selectively form duplex molecules with complementarystretches of DNA fragments. Depending on the application envisioned, onewill desire to employ varying conditions of hybridization to achievevarying degrees of selectivity of probe towards target sequence. Forapplications requiring high selectivity, one will typically desire toemploy relatively stringent conditions to form the hybrids, e.g., onewill select relatively low salt and/or high temperature conditions, suchas provided by about 0.02 M to about 0.15 M NaCl at temperatures ofabout 50° C. to about 70° C. A stringent conditions, for example, is towash the hybridization filter at least twice with high-stringency washbuffer (0.2×SSC, 0.1% SDS, 65° C.). Appropriate stringency conditionswhich promote DNA hybridization, for example, 6.0× sodiumchloride/sodium citrate (SSC) at about 45° C., followed by a wash of2.0×SSC at 50° C., are known to those skilled in the art or can be foundin —Current Protocols In Molecular Biology, John Wiley & Sons, N.Y.(1989), 6.3.1-6.3.6. For example, the salt concentration in the washstep can be selected from a low stringency of about 2.0×SSC at 50° C. toa high stringency of about 0.2×SSC at 50° C. In addition, thetemperature in the wash step can be increased from low stringencyconditions at room temperature, about 22° C., to high stringencyconditions at about 65° C. Both temperature and salt may be varied, oreither the temperature or the salt concentration may be held constantwhile the other variable is changed. Such selective conditions toleratelittle, if any, mismatch between the probe and the template or targetstrand. Detection of DNA sequences via hybridization is well-known tothose of skill in the art, and the teachings of U.S. Pat. Nos. 4,965,188and 5,176,995 are exemplary of the methods of hybridization analyses.

[0031] In a particularly preferred embodiment, a nucleic acid of thepresent invention will specifically hybridize to one or more of thenucleic acid molecules set forth in SEQ ID NO:5 and SEQ ID NO:6 orcomplements or fragments of either under high stringency conditions. Inone aspect of the present invention, a marker nucleic acid molecule ofthe present invention has the nucleic acid sequence set forth in SEQ IDNO:5 or SEQ ID NO:6 or complements thereof or fragments of either.

[0032] In another aspect of the present invention, a marker nucleic acidmolecule of the present invention shares between 80% and 100% or 90% and100% sequence identity with the nucleic acid sequence set forth in SEQID NO: 5 and 6 or complement thereof or fragments of either. In afurther aspect of the present invention, a marker nucleic acid moleculeof the present invention shares between 95% and 100% sequence identitywith the sequence set forth in SEQ ID NO:5 and SEQ ID NO:6 or complementthereof or fragments of either. SEQ ID NO:5 and SEQ ID NO:6 may be usedas markers in plant breeding methods to identify the progeny of geneticcrosses similar to the methods described for simple sequence repeat DNAmarker analysis, in “DNA markers: Protocols, applications, andoverviews: (1997) 173-185, Cregan, et al., eds., Wiley-Liss NY; hereinincorporated by reference in its entirely. The hybridization of theprobe to the target DNA molecule can be detected by any number ofmethods known to those skilled in the art, these can include, but arenot limited to, fluorescent tags, radioactive tags, antibody based tags,and chemiluminescent tags.

[0033] Regarding the amplification of a target nucleic acid sequence(e.g., by PCR) using a particular amplification primer pair, “stringentconditions” are conditions that permit the primer pair to hybridize onlyto the target nucleic-acid sequence to which a primer having thecorresponding wild-type sequence (or its complement) would bind andpreferably to produce a unique amplification product, the amplicon.

[0034] The term “specific for (a target sequence)” indicates that aprobe or primer hybridizes under stringent hybridization conditions onlyto the target sequence in a sample comprising the target sequence.

[0035] As used herein, “amplified DNA” or “amplicon” refers to theproduct of nucleic-acid amplification of a target nucleic acid sequencethat is part of a nucleic acid template. For example, to determinewhether the cotton plant resulting from a sexual cross containstransgenic event genomic DNA from the cotton plant of the presentinvention, DNA extracted from a cotton plant tissue sample may besubjected to nucleic acid amplification method using a primer pair thatincludes a primer derived from flanking sequence in the genome of theplant adjacent to the insertion site of inserted heterologous DNA, and asecond primer derived from the inserted heterologous DNA to produce anamplicon that is diagnostic for the presence of the event DNA. Theamplicon is of a length and has a sequence that is also diagnostic forthe event. The amplicon may range in length from the combined length ofthe primer pairs plus one nucleotide base pair, preferably plus aboutfifty nucleotide base pairs, more preferably plus about two hundrednucleotide base pairs, more preferably plus about three hundrednucleotide base pairs, and even more preferably plus about five hundrednucleotide base pairs. Alternatively, a primer pair can be derived fromflanking sequence on both sides of the inserted DNA so as to produce anamplicon that includes the entire insert nucleotide sequence. A memberof a primer pair derived from the plant genomic sequence may be locateda distance from the inserted DNA sequence, this distance can range fromone nucleotide base pair up to about twenty thousand nucleotide basepairs. The use of the term “amplicon” specifically excludes primerdimers that may be formed in the DNA thermal amplification reaction.

[0036] Nucleic-acid amplification can be accomplished by any of thevarious nucleic-acid amplification methods known in the art, includingthe polymerase chain reaction (PCR). A variety of amplification methodsare known In the art and are described, inter alia, in U.S. Pat. No.4,683,195, herein incorporated by reference in its entirety and in U.S.Pat. No. 4,683,202, herein incorporated by reference in its entirety,and in PCR Protocols: A Guide to Methods and Applications, ed. Innis etal., Academic Press, San Diego, 1990. PCR amplification methods havebeen developed to amplify up to 22 kb of genomic DNA and up to 42 kb ofbacteriophage DNA (Cheng et al., Proc. Natl. Acad. Sci. USA91:5695-5699, 1994, herein incorporated by reference in its entirety).These methods as well as other methods known in the art of DNAamplification may be used in the practice of the present invention. Thesequence of the heterologous transgene DNA insert or flanking genomicsequence from cotton event PV-GHGT07(1445) can be verified (andcorrected if necessary) by amplifying such sequences from the eventusing primers derived from the sequences provided herein followed bystandard DNA sequencing of the PCR amplicon or of the cloned DNA.

[0037] The amplicon produced by these methods may be detected by aplurality of techniques. Agarose gel electrophoresis and staining withethidium bromide is a common well known method of detecting DNAamplicons. Another such method is Genetic Bit Analysis (Nikiforov, etal. Nucleic Acid Res. 22:4167-4175, 1994) where an DNA oligonucleotideis designed which overlaps both the adjacent flanking genomic DNAsequence and the inserted DNA sequence. The oligonucleotide isimmobilized in wells of a microwell plate. Following PCR of the regionof interest (using one primer in the inserted sequence and one in theadjacent flanking genomic sequence), a single-stranded PCR product canbe hybridized to the immobilized oligonucleotide and serve as a templatefor a single base extension reaction using a DNA polymerase and labelledddNTPs specific for the expected next base. Readout may be fluorescentor ELISA-based. A signal indicates presence of the insert/flankingsequence due to successful amplification, hybridization, and single baseextension.

[0038] Another method is the Pyrosequencing technique as described byWinge (Innov. Pharma. Tech. 00:18-24, 2000). In this method anoligonucleotide is designed that overlaps the adjacent genomic DNA andinsert DNA junction. The oligonucleotide is hybridized tosingle-stranded PCR product from the region of interest (one primer inthe inserted sequence and one in the flanking genomic sequence) andincubated in the presence of a DNA polymerase, ATP, sulfurylase,luciferase, apyrase, adenosine 5″ phosphosulfate and luciferin. DNTPsare added individually and the incorporation results in a light signalwhich is measured. A light signal indicates the presence of thetransgene insert/flanking sequence due to successful amplification,hybridization, and single or multi-base extension.

[0039] Fluorescence Polarization as described by Chen et al., (GenomeRes. 9:492-498, 1999) is a method that can be used to detect theamplicon of the present invention. Using this method an oligonucleotideis designed which overlaps the genomic flanking and inserted DNAjunction. The oligonucleotide is hybridized to single-stranded PCRproduct from the region of interest (one primer in the inserted DNA andone in the flanking genomic DNA sequence) and incubated in the presenceof a DNA polymerase and a fluorescent-labeled ddNTP. Single baseextension results in incorporation of the ddNTP. Incorporation can bemeasured as a change in polarization using a fluorometer. A change inpolarization indicates the presence of the transgene insert/flankingsequence due to successful amplification, hybridization, and single baseextension.

[0040] Taqman (PE Applied Biosystems, Foster City, Calif.) is describedas a method of detecting and quantifying the presence of a DNA sequenceand is fully understood in the instructions provided by themanufacturer. Briefly, a FRET oligonucleotide probe is designed whichoverlaps the genomic flanking and insert DNA junction. The FRET probeand PCR primers (one primer in the insert DNA sequence and one in theflanking genomic sequence) are cycled in the presence of a thermostablepolymerase and dNTPs. Hybridization of the FRET probe results incleavage and release of the fluorescent moiety away from the quenchingmoiety on the FRET probe. A fluorescent signal indicates the presence ofthe flanking/transgene insert sequence due to successful amplificationand hybridization.

[0041] Molecular Beacons have been described for use in sequencedetection as described in Tyangi et al. (Nature Biotech. 14:303-308,1996) Briefly, a FRET oligonucleotide probe is designed that overlapsthe flanking genomic and insert DNA junction. The unique structure ofthe FRET probe results in it containing secondary structure that keepsthe fluorescent and quenching moieties in close proximity. The FRETprobe and PCR primers (one primer in the insert DNA sequence and one inthe flanking genomic sequence) are cycled in the presence of athermostable polymerase and dNTPs. Following successful PCRamplification, hybridization of the FRET probe to the target sequenceresults in the removal of the probe secondary structure and spatialseparation of the fluorescent and quenching moieties. A fluorescentsignal results. A fluorescent signal indicates the presence of theflanking genomic/transgene insert sequence due to successfulamplification and hybridization.

[0042] DNA detection kits can be developed using the compositionsdisclosed herein and the methods well known in the art of DNA detection.The kits are useful for identification of cotton event PV-GHGT07(1445)DNA in a sample and can be applied to methods for breeding cotton plantscontaining PV-GHGT07(1445) DNA. The kits contain DNA sequenceshomologous or complementary to SEQ ID NO:7 or SEQ ID NO:8 or to DNAsequences homologous or complementary to DNA contained in the transgenegenetic elements of PV-GHGT07(1445) DNA, these DNA sequences can be usedin DNA amplification reactions or as probes in a DNA hybridizationmethod. The kits may also contain the reagents and materials necessaryfor the performance of the detection method.

[0043] The transgene genetic element DNA molecules contained inPV-GHGT07(1445) consists of the cauliflower mosaic virus 35S promoter(P-CaMV. 35S, U.S. Pat. No. 5,352,605), herein incorporated by referencein its entirety; operably connected to the neomycin phosphotransferasegene (nptII) (Fraley et al. Proc Natl. Acad Sci USA 80:4803-4807, 1983,herein incorporated by reference in its entirety); operably connected tothe nopaline synthase 3” termination region (Fraley et al. Proc Natl.Acad Sci USA 80:4803-4807, 1983, herein incorporated by reference in itsentirety); the Figwort mosaic promoter (U.S. Pat. No. 5,378,619, hereinincorporated by reference in its entirety); operably connected to anArabidopsis EPSPS chloroplast transit peptide (TS-At.EPSPS:CTP2, Klee etal., Mol. Gen. Genet. 210:47-442,1987, herein incorporated by referencein its entirety); operably connected to a glyphosate tolerant5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS) from Agrobacterlumsp. strain CP4 (AGRTU.aroA:CP4 EPSPS, U.S. Pat. No. 5,633,435, hereinincorporated by reference in its entirety); operably connected to the 3″termination region from pea ribulose 1,5-bisphosphate carboxylase E9(Coruzzi, et al., EMBO J. 3:1671-1679, herein incorporated by referencein its entirety). The DNA polynucleotide sequences or fragments thereofdisclosed in these references can be used as DNA primers or probes inthe methods of the present invention.

[0044] The following examples are included to demonstrate examples ofcertain preferred embodiments of the invention. It should be appreciatedby those of skill in the art that the techniques disclosed in theexamples that follow represent approaches the inventors have foundfunction well in the practice of the invention, and thus can beconsidered to constitute examples of preferred modes for its practice.However, those of skill in the art should, in light of the presentdisclosure, appreciate that many changes can be made in the specificembodiments that are disclosed and still obtain a like or similar resultwithout departing from the spirit and scope of the invention.

EXAMPLES EXAMPLE 1

[0045] DNA from PV-GHGT07(1445) Roundup Ready® cotton event wasextracted from cotton leaves containing the PV-GHGT07(1445) event andnontransgenic cotton line Coker 312. Young leaves were ground in liquidnitrogen and the total DNA extracted following a method modified fromFulton et al. Pl. Mol. Biol. Rep. 13:207-209, 1995, herein incorporatedby reference in its entirety. Approximately 1 gram of the ground leaftissue was transferred to 13 milliliters (ml) centrifuge tube containing6 ml of the extraction buffer [2.5 ml DNA extraction buffer (350 mMSorbitol, 100 mM Tris pH 7.5, 5 mM EDTA), 2.5 ml nuclei lysis buffer(200 mM Tris pH 7.5, 50 mM EDTA, 2 M NaCl, 2% CTAB), and 1 ml Sarkosyl(5% solution)]. The samples were incubated at 65° C. for approximately30 minutes with intermittent mixing. Four and a half milliliters of amixture of chloroform:isoamyl alcohol (24:1) was added to the samples.The suspension was mixed for 2 to 3 minutes, and the two phasesseparated by centrifugation for 15 minutes at ˜2700 rpm at 4° C. Theaqueous (top) layer was removed using a transfer pipette and placed intoa 13 ml centrifuge tube. Five milliliters of 100% isopropanol wereadded, and the tubes were mixed by inversion to precipitate the DNA. Theprecipitated DNA was pelleted by centrifuging at ˜2700 rpm for 5 minutesat 4° C. The pellet was washed with approximately 1 ml of 70% ethanoland centrifuged for an additional 5 minutes at ˜2700 rpm at 4° C. TheDNA was resuspended in 0.25-0.5 ml TE, pH 8.0, and stored in a 4° C.refrigerator.

[0046] The DNA extracted from the cotton leaf tissue was used in a PCRDNA amplification of the 5′ genomic/transgene insert sequences usingprimer 1 (SEQ ID NO:1, 5′ TGCGATACTAGGCTTTTGGTTTCTT 3′) and primer 2(SEQ ID NO:2, 5′ AGTTATACTCATGGATTTGTAGTTGAG 3′), and the 3′genomic/transgene insert sequences flanking using primer 3 (SEQ ID NO:3,5′ AGGCATCTTGAACGATAGCCTTTC 3′) and primer 4 (SEQ ID NO:4, 5′AACACCTAATACAAGTCATACATACA 3′). The PCR DNA amplification analyses wereconducted using genomic DNA extracted from cotton event PV-GHGT07(1445)and non-transgenic cotton line Coker 312. The amplification reaction forthe 5′ flanking genomic sequence was conducted using Supermix from GibcoBRL (Gaithersburg, Md.) with a final concentration of 0.4 μM for Primer1 and Primer 2 in a 50 μI reaction volume. The PCR for the 3′ flankinggenomic sequence was conducted in a 50 μl reaction volume containing afinal concentration of 1.5 mM Mg²⁺, 200 μM each dNTP, 2 units of Taq DNApolymerase, and 0.4 μM of Primer 3 and Primer 4. The reactions wereperformed under the following cycling conditions: 1 cycle at 94° C. for1 minute; 30 cycles of 96° C. for 30 seconds, 50° C. for 30 seconds, 72°C. for 45 seconds; 1 cycle at 72° C. for 5 minutes. The PCR productswere separated using 2.0% agarose gel electrophoresis at 100 V for ˜1hour and visualized by ethidium bromide staining. PCR products of theexpected sizes representing the 5′ and 3′ flanking sequences wereisolated by separation of the PCR products on a 2.0% agarose gel byelectrophoresis. PCR products, representing 5′ regions that span thejunction between the PV-GHGT07(1445) transgenic insertion and theneighboring flanking cotton genomic DNA sequence were purified byagarose gel electrophoresis followed by isolation from the agarosematrix using the QlAquick Gel Extraction Kit (catalog # 28704, QiagenInc., Valencia, Calif.). The purified PCR products were then sequencedwith by DNA sequence analysis (ABI Prism™ 377, PE Biosystems, FosterCity, Calif. and DNASTAR sequence analysis software, DNASTAR Inc.,Madison, Wis.).

[0047] A portion of the 5′ PCR product DNA sequence was determined,resulting in a 320 nucleotide base pair sequence representing the 5′genomic/transgene insert sequence of cotton PV-GHGT07(1445) andidentified as SEQ ID NO:7. A portion of the 3′ PCR product DNA sequencewas determined resulting in a 499 nucleotide base pair sequencerepresenting the 3′ genomic/transgene insert sequence of cottonPV-GHGT07(1445) and identified in SEQ ID NO:8.

[0048] The genomic/transgene junction sequences, SEQ ID NO:5 and SEQ IDNO:6 are novel DNA sequences in PV-GHGT07(1445) that are diagnostic forcotton plant PV-GHGT07(1445) and its progeny. SEQ ID NO:5 and SEQ IDNO:6 represent 9 nucleotides on each side of an insertion site of atransgene sequence fragment and the cotton genome. Junction sequence SEQID NO:5 is found at nucleotide positions 164-181 of SEQ ID NO:7, andjunction sequence SEQ ID NO:6, is located at nucleotide positions366-383 of SEQ ID NO:8, representing the genomic/transgene junctionsequences of the transgene insert with cotton genomic DNA sequence.

EXAMPLE 2

[0049] DNA event primer pairs are used to produce an amplicon diagnosticfor PV-GHGT07(1445). These event primer pairs include, but are notlimited to SEQ ID NO: 9 and SEQ ID NO: 10 that when used in a DNAamplification method (PCR) produce an amplicon of about 1107 nucleotidebase pairs (bp). In addition to these primer pairs, any primer pairderived from the amplicon product of SEQ ID NO:9 and SEQ ID NO:10, orSEQ ID NO:7, or SEQ ID NO:8 that in a DNA amplification reactionproduces an amplicon diagnostic for event 1445 is an aspect of thepresent invention. The amplification conditions for this analysis areillustrated in Table 1 and Table 2, however, any modification of thesemethods that use DNA primers to produce an amplicon diagnostic forPV-GHGT07(1445) is within the ordinary skill of the art. The analysis ofPV-GHGT07(1445) plant tissue sample should include a positive tissuecontrol from PV-GHGT07(1445), a negative control from a cotton plantthat is not PV-GHGT07(1445), and a negative control that contains notemplate cotton DNA. Additional primer sequences can be selected fromSEQ ID NO:7 and SEQ ID NO:8 by those skilled in the art of DNAamplification methods, and conditions optimized for the production of anamplicon that may differ from the methods shown in Table 1 and Table 2,but result in an amplicon diagnostic for PV-GHGT07(1445). The use ofthese DNA primer sequences with modifications to the methods of Table 1and 2 are within the scope of the invention. The amplicon produced bythe use at least one primer sequence derived from SEQ ID NO:7 and SEQ IDNO:8 that is diagnostic for PV-GHGT07(1445) can be used in the describedmethods and is an aspect of the invention. The assay for thePV-GHGT07(1445) amplicon can be performed by using a StratageneRobocycler, MJ Engine, Perkin-Elmer 9700, or Eppendorf MastercyclerGradient thermocycler as shown in Table 2, or by methods and apparatusknown to those skilled in the art. TABLE 1 PCR procedure and reactionmixture for the confirmation of 1445 5′ transgene insert/genomicjunction region. Step Reagent Amount Comments 1 Nuclease-free water addto final volume of 50 μl 2 10 × reaction buffer 5.0 μl 1 × final (withMgCl₂) concentration of buffer, 1.5 mM final concentration of MgCl₂ 3 10mM solution of dATP, dCTP,   1 μl 200 μM final dGTP, and dTTPconcentration of each dNTP 4 event primer 9 (SEQ ID NO 9)   1 μl 0.2 μMfinal (resuspended in 1 × TE buffer or concentration nuclease free waterto a concentration of 10 μM) 5 event primer 10 (SEQ ID NO 10)   1 μl 0.2μM final (resuspended in 1 × TE buffer or concentration nuclease freewater to a concentration of 10 μM) 6 REDTaq DNA polymerase 0.5 μl(recommended to switch 1 uart/reaction (1 unit/μl) pipets prior to nextstep). 8 Extracted DNA (template) Samples to be analyzed individualleaves 50-200 ng of genomic DNA pooled leaves 200 ng of genomic DNANegative control 100 ng of cotton genomic DNA (not 1445) Negativecontrol no DNA template solution Positive control 50-200 ng of 1445genomic DNA

[0050] TABLE 2 Suggested PCR parameters for different thermocyclersGently mix and, if needed (no hot top on thermocycler), add 1-2 drops ofmineral oil on top of each reaction. Proceed with the PCR in aStratagene Robocycler, MJ Engine Perkin Elmer 9700, or EppendorfMastercycler Gradient thermocycler using the following cyclingparameters. The MJ Engine or Eppendorf Mastercycler Gradientthermocycler should be run in the calculated mode. Run the Perkin Elmer9700 thermocycler with the ramp speed set at maximum. Cycle No.Settings: Stratagene Robocycler  1 94° C. 3 minutes 30 96° C. 1 minute60° C. 1 minute 72° C. 1 minute  1 72° C. 5 minutes Cycle No. Settings:MJ Engine or Perkin Elmer 9700  1 94° C. 4 minutes 40 94° C. 1 minute64° C. 30 seconds 68° C. 3 minute  1 72° C. 5 minutes Cycle No.Settings: Eppendorf Mastercycler Gradient  1 94° C. 3 minutes 30 96° C.15 seconds 60° C. 15 seconds 72° C. 1 minute  1 72° C. 5 minutes

EXAMPLE 3

[0051] The methods used to identify heterozygous from homozygous cottonprogeny containing event PV-GHGT07(1445) are described in the zygosityassay in Table 3 and Table 4. The DNA primers used in the zygosity assayare primer 9,5′ GATCCATCCCATAGGGTCGATC 3′, (SEQ ID NO:9) primer 11, 5′CCAAGGCAATTACCTTACTGCC 3′, (SEQ ID NO:11) and primer 12, 5′TTAAAAGACAGGTTAGCGGTGGC 3′. (SEQ ID NO:12)

[0052] SEQ ID NO:9, SEQ ID NO:11 and SEQ ID NO:12 when used in thesereaction methods produce a DNA amplicon of about 455 bp fornon-transgenic cotton, two DNA amplicons of about 455 bp and about 184bp for heterozygous cotton containing event PV-GHGT07(1445), and a DNAamplicon of about 184 bp for homozygous cotton containing eventPV-GHGT07(1445). The controls for this analysis should include apositive control from homozygous and heterozygous cotton containingevent PV-GHGT07(1445), a negative control from non-transgenic cotton,and a negative control that contains no template DNA. This assay isoptimized for use with a Stratagene Robocycler, MJ Engine, Perkin-Elmer9700, or Eppendorf Mastercycler Gradient thermocycler. Other methods andapparatus known to those skilled in the art that produce amplicons thatidentify the zygosity of the progeny of crosses made withPV-GHGT07(1445) event cotton plants is within the skill of the art.TABLE 3 Zygosity assay reaction solutions Step Reagent Amount Comments 1Nuclease-free water add to 20 μl final volume 2 10X reaction buffer(with MgCl₂)   2 μl 1.5 mM final concentration of MgCl₂ 3 10 mM solutionof dATP, dCTP, 0.4 μl 200 μM final dGTP, and dTTP concentration of eachdNTP 4 Primer 9 (resuspended in 1 × TE 0.5 μl 0.25 μM final buffer ornuclease-free water to a concentration concentration of 10 μM) 5 Primer11 (resuspended in 1 × TE 0.8 μl 0.4 μM final buffer or nuclease-freewater to a concentration concentration of 10 μM) 6 Primer 12(resuspended in 1 × TE 0.3 μl 0.15 μM final buffer or nuclease-freewater to a concentration concentration of 10 μM) 7 REDTaq DNA polymerase1.0 μl (recommended to 1 unit/reaction (1 unit/μl) switch pipets priorto next step). 8 Extracted DNA (template) Samples to be analyzed 4.80 ngof genomic DNA (individual leaves) Negative control 4 ng ofnon-transgenic cotton genomic DNA Negative control no DNA template(solution in which DNA was resuspended) Positive control 4 ng of genomicDNA from known event 1445 heterorygous cotton Positive control 4 ng ofgenomic DNA from known event 1445 homorygous cotton

[0053] TABLE 4 Zygosity assay thermocycler conditions Gently mix and ifneeded (no hot top on thermocycler), add 1-2 drops of mineral oil on topof each reaction. Proceed with the PCR in a Stratagene Robocycler, MJEngine, Perkin-Elmer 9700 or Eppendorf Mastercycler Gradientthermocycler using the following cycling parameters. When running thePCR in the Eppendorf Mastercycler Gradient or MI Engine, thethermocycler should be run in the calculated mode. When running the PCRin the Perkin-Elmer 9700, run the thermocycler with the ramp speed setat maximum. Cycle No. Settings: Stratagene Robocycler  1 94° C. 3minutes 18 94° C. 1 minute 60° C. 1 minute 72° C. 1 minute and 30seconds  1 72° C. 10 minutes Cycle No. Settings: MJ Engine orPerkin-Elmer 9700  1 94° C. 3 minutes 38 94° C. 30 seconds 60° C. 30seconds 72° C. 1 minute and 30 seconds  1 72° C. 10 minutes Cycle No.Settings: Eppendorf Mastercycler Gradient  1 94° C. 3 minutes 38 94° C.15 seconds 60° C. 15 seconds 72° C. 1 minute and 30 seconds  1 72° C. 10minutes

[0054] Having illustrated and described the principles of the presentinvention, it should be apparent to persons skilled in the art that theinvention can be modified in arrangement and detail without departingfrom such principles. We claim all modifications that are within thespirit and scope of the appended claims.

1 12 1 25 DNA Gossypium hirsutum 1 tgcgatacta ggcttttggt ttctt 25 2 27DNA Pisum sativum 2 agttatactc atggatttgt agttgag 27 3 24 DNAAgrobacterium tumefaciens 3 aggcatcttg aacgatagcc tttc 24 4 26 DNAGossypium hirsutum 4 aacacctaat acaagtcata cataca 26 5 18 DNA ArtificialSequence misc_feature (1)..(18) artificial DNA sequence, part cottongenome and part transgene 5 cgattcagat caaacact 18 6 18 DNA ArtificialSequence misc_feature (1)..(18) artificial DNA sequence, part cottongenome and part transgene 6 caaatgtcaa tagcttgg 18 7 320 DNA ArtificialSequence misc_feature (1)..(320) artificial DNA sequence, part cottongenome and part transgene 7 ttgcgatact aggcttttgg tttctttggt ttatgtgatatttggtatta ttttattcaa 60 atacggtggc taacataagt agctgtgagt gagatgatcccagtaatgtc taaaatcacg 120 gagcataaac ttaataaata taattatctt gattggagtaagacgattca gatcaaacac 180 tgatagttta aactgaaggc gggaaacgac aatctgatcccagcttgggc tgcaggtcga 240 ttgatgcatg ttgtcaatca attggcaagt cataaaatgcattaaaaaat attttcatac 300 tcaactacaa atccatgagt 320 8 499 DNA ArtificialSequence misc_feature (1)..(499) artificial DNA sequence, part cottongenome and part transgene 8 ggcatttgta ggtgccacct tccttttcta ctgtccttttgatgaagtga caggtaggat 60 cggaaagcta gcttggctgc catttttggg gtgaggccgttcgcggccga ggggcgccag 120 cccctggggg gatgggaggc ccgcgttagc gggccgggagggttcgagaa gggggggcac 180 cccccttcgg cgtgcgcggt cacgcgcaca gggcgcagccctggttaaaa acaaggttta 240 taaatattgg tttaaaagca ggttaaaaga caggttagcggtggccgaaa aacgggcgga 300 aacccttgca aatgctggat tttctgcctg tggacagcccctcaaatgtc aataggtgcg 360 cccctcaaat gtcaatagct tggctgagaa atgatgcatgacttttggag atctaaagct 420 ttattggcag taaggtaatt gccttggcta accactttaaatttgttaaa gaattaattg 480 tttacttgga attttgtat 499 9 22 DNA artificialsequence misc_feature (1)..(22) synthetic primer sequence 9 gatccatcccatagggtcga tc 22 10 24 DNA artificial sequence misc_feature (1)..(24)synthetic primer sequence 10 ctaagatcga actctccgac acta 24 11 22 DNAartificial sequence misc_feature (1)..(22) synthetic primer sequence 11ccaaggcaat taccttactg cc 22 12 23 DNA artificial sequence misc_feature(1)..(23) synthetic primer sequence 12 ttaaaagaca ggttagcggt ggc 23

1. A DNA molecule isolated from cotton tissue identified as SEQ ID NO:7.2. A primer pair of DNA molecules comprising a sufficient length ofcontiguous nucleotides of SEQ ID NO:7 or complements thereof wherein afirst DNA molecule of the primer pair resides in a transgene insert DNAsequence of SEQ ID NO:7 and a second DNA molecule of the primer pairresides in the cotton genomic DNA sequence of SEQ ID NO:7 and the pairof DNA molecules are useful as DNA nucleotide primers in a DNAamplification method.
 3. A DNA molecule isolated from cotton tissueidentified as SEQ ID NO:8.
 4. A primer pair of DNA molecules comprisinga sufficient length of contiguous nucleotides of SEQ ID NO:8 orcomplements thereof wherein a first DNA molecule of the primer pairresides in a transgene insert DNA sequence of SEQ ID NO:8 and a secondDNA molecule of the primer pair resides in the cotton genomic DNAsequence of SEQ ID NO:8 and the pair of DNA molecules are useful as DNAnucleotide primers in a DNA amplification method.
 5. A method ofdetecting the presence of DNA corresponding to the genomic/transgene DNAof cotton event PV-GHGT07(1445) event in a sample, the methodcomprising: (a) contacting the sample comprising cotton DNA with aprimer pair of claim 2, that when used in a nucleic-acid amplificationreaction with DNA from cotton event PV-GHGT07(1445), produces anamplicon that is diagnostic for cotton event PV-GHGT07(1445); and (b)performing a nucleic acid amplification reaction, thereby producing theamplicon; and (c) detecting the amplicon.
 6. An isolated DNA moleculecomprising the amplicon produced by the method of claim
 5. 7. A DNAdetection kit specific for genomic/transgene DNA of cotton eventPV-GHGT07(1445) and its progeny comprising at least one DNA molecule ofsufficient length of contiguous DNA polynucleotides to function in a DNAdetection method, that is homologous or complementary to SEQ ID NO:7. 8.A method of detecting the presence of DNA corresponding to thegenomic/transgene. DNA of cotton event PV-GHGT07(1445) event in asample, the method comprising: (a) contacting the sample comprisingcotton DNA with a primer pair of claim 4, that when used in anucleic-acid amplification reaction with DNA from cotton eventPV-GHGT07(1445), produces an amplicon that is diagnostic for cottonevent PV-GHGT07(1445); and (b) performing a nucleic acid amplificationreaction, thereby producing the amplicon; and (c) detecting theamplicon.
 9. An isolated DNA molecule comprising the amplicon producedby the method of claim
 8. 10. A DNA detection kit specific forgenomic/transgene DNA of cotton event PV-GHGT07(1445) and its progenycomprising at least one DNA molecule of sufficient length of contiguousDNA polynucleotides to function in a DNA detection method, that ishomologous or complementary to SEQ ID NO:8.
 11. A method of detectingthe presence of a genomic/transgene DNA corresponding to thePV-GHGT07(1445) event in a sample, the method comprising: (a) contactingthe sample comprising cotton DNA with a polynucleotide probe thathybridizes under stringent hybridization conditions with DNA from cottonevent PV-GHGT07(1445) and does not hybridize under the stringenthybridization conditions with a non PV-GHGT07(1445) cotton plant DNA;and (b) subjecting the sample and probe to stringent hybridizationconditions; (c) detecting hybridization of the probe to the DNA.
 12. Anisolated DNA molecule comprising a genomic/transgene DNA junctionsequence of cotton event PV-GHGT07(1445) identified as SEQ ID NO:5 orDNA molecules substantially homologous to said DNA molecule orcomplements thereof.
 13. An isolated DNA molecule comprising agenomic/transgene DNA junction sequence of cotton event PV-GHGT07(1445)identified as SEQ ID NO:6 or DNA molecules substantially homologous tosaid DNA molecule or complements thereof.
 14. A method of breeding acotton plant comprising a glyphosate tolerant trait that is geneticallylinked to a complement of a marker nucleic acid, wherein said markernucleic acid molecule is SEQ ID NO:5 or SEQ ID NO:6 or complementsthereof.
 15. A method of determining the genomic/transgene DNA zygosityof the progeny of cotton Plant PV-GHGT07(1445) comprising: (a)contacting the sample comprising cotton DNA with a primer set comprisingSEQ ID NO:9, SEQ ID NO:11 and SEQ ID NO:12, that when used in anucleic-acid amplification reaction with genomic DNA from cotton eventPV-GHGT07(1445), produces a first amplicon that is diagnostic for cottonevent PV-GHGT07(1445); and (b) performing a nucleic acid amplificationreaction, thereby producing the first amplicon; and (c) detecting thefirst amplicon; and (d) contacting the sample comprising cotton DNA withsaid primer set, that when used in a nucleic-acid amplification reactionwith genomic DNA from cotton plants produces a second ampliconcomprising the native cotton genomic DNA homologous to the cottongenomic region of a transgene insertion identified as cotton eventPV-GHGT07(1445); and (e) performing a nucleic acid amplificationreaction, thereby producing the second amplicon; and (f) detecting thesecond amplicon; and (g) comparing the first and second amplicons in asample, wherein the presence of both amplicons indicates the sample isheterozygous for the transgene insertion.
 16. An isolated DNA nucleotideprimer sequence comprising SEQ ID NO:9 or its complement.
 17. Anisolated DNA nucleotide primer sequence comprising SEQ ID NO:10 or itscomplement.
 18. An isolated DNA nucleotide primer sequence comprisingSEQ ID NO:11 or its complement.
 19. An isolated DNA nucleotide primersequence comprising SEQ ID NO:12 or its complement.
 20. An isolated DNAmolecule comprising the first amplicon produced by the method of claim15.
 21. An isolated DNA molecule comprising the second amplicon producedby the method of claim 15.